Team:TU Darmstadt/Notebook/sec1/K1602005

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K1602005 - Inducible Itaconic Acid Producing Construct (T7-B0034-cadA)

Figure 1 cPCR of psB1A2-RBS-cadA (X). The size of the amplified product was around 1.8 kbp. DNA marker: 2-Log DNA Ladder (NEB).

To include the RBS-cadA brick into an IPTG inducable vector plasmid DNA containing RBS-cadA was isolated and cut with XbaI and PstI. The psB1A2 vector containing the T7 promoter was cut with SpeI and PstI and afterwards dephosphorylated. Both restriction products were then ligated with T4-ligase and transformed into Top10 E.coli by heat shock transformation. Afterwards the cells were spread out on an AMP agar plate. Clones were screened with colony PCR using VF2 and VR oligonucleotides. Plasmid DNA was isolated from positive clones which were verified by Sanger sequencing. Afterwards the psB1A2 vector containing the brick (RBS-cadA) was cut with EcoRI and PstI as well as a psB1C3. Both restriction products were then ligated with T4-ligase.